This invention concerns a process for the production of the sodium salt of high molecular weight hyaluronic acid by large-scale fermentation of a microorganism of the genus Streptococcus.
Hyaluronic acid is a naturally occurring glycosaminoglycan consisting of a linear polymer of molecular weight of 50,000-13,000,000 daltons. It is a polysaccharide made of a repeating units of glucuronic acid and N-acetyl-glucosamine, bound by alternating .beta.1-3 and .beta.1-4 bonds.
Hyaluronic acid is present in various connective tissues of animals, such as skin and cartilage. Some organs are specifically rich in hyaluronic acid, such as the umbilical cord, synovial fluid, the vitreous humor and rooster combs. In addition, hyaluronic acid is produced by various microorganisms, such as streptococci Type A and C.
In skin and cartilage, the role of hyaluronic acid is to bind water and retain the tonicity and elasticity of the tissue. In joint fluids, the viscous hyaluronic acid solution serves as a lubricant to provide a protective environment to the cells. A solution of ultrapure hyaluronic acid from rooster combs has been in use for several years as a supportive medium in opthalmic surgery, see U.S. Pat. No. 4, 141,973 of E. A. Balazs (1979). A similar preparation has been shown to be beneficial in the treatment of inflamed knee joints of race horses. Another use of hyaluronic acid results from its highly hydrophilic nature, making it an ideal constitutent of moisturization lotions for cosmetic use, U.S. Pat. No. 4, 303,676 of E. Balazs (1981).
Hyaluronic acid has been isolated from the various biological sources, as described above, including microbial broth. The isolation and characterization of hyaluronic acid has been described by Meyer et al., J. Biol. Chem. 107,629 (1934); J. Biol. Chem. 114,689 (1936), and has recently been reviewed in Methods in Enzymol. 28, 73 (1972). The structure of hyaluronic acid was elucidated by Weissman et al., J. Am. Chem. Soc. 76, 1753 (1954) and Meyer, Fed. Proc. 17, 1075 (1958).
The production of hyaluronic acid by Streptococcus was first shown by Forrest et al., J. Biol. Chem. 118, 61 (1937), and further elaborated on since by various researchers, such as Roseman et al., J. Biol. Chem 203,213 (1953), Pierce and White, Proc. Soc. Exp. Biol. Med. 87, 50 (1954), U.S. Pat. No. 2,975,104 of G. H. Warren (1961), and Sunghara et al., J. Biol. Chem. 254, 6252 (1979), demonstrating the identity of hyaluronic acid from animal and microbial sources. Procedures have been published for batch fermentations of Type A streptococci and hyaluronic acid isolation on small to medium scales Thonard et al., J. Biol. Chem. 239, 726 (1964); Holmstrom and Ricica, Appl. Microbio. 15, 1409 (1967); Kjems and Lebech, Acta Path. Microbiol. Scand. 84, 162 (1976). These procedures included anaerobic fermentations of the pathogenic bacteria, and resulted in yields of 0.4-1 grams/liter of hyaluronic acid of a molecular weight of 700,000 or less.
Other procedures have concerned the aerobic fermentation of streptococci to produce hyaluronic acid such as Japanese Pat. Publication Kokai No. 58-056692, published Apr. 4, 1983, by inventors, Akasaka H, et al. Other publications such as, U.S. Pat. No. 4,141,973, Feb. 27, 1979 by E. A. Balazs, concerned the production and purification of hyaluronic acid from sources such as animal connective tissue. The hyaluronic acid production and purification procedures disclosed in the prior art did not, however, yield hyaluronic acid of an average molecular weight of greater than 2.0.times.10.sup.6 daltons. This is largely due to the fact that hyaluronic acid is easily degraded by shearing or oxidized in reactions catalyzed by impurities or metal ions present in the hyaluronic acid composition.
The novel process described herein results in hyaluronic acid of a molecular weight from about 1.times.10.sup.6 to about 4.0.times.10.sup.6 daltons, in a yield of about 2 grams/liter in anaerobic fermentation and about 4-6 grams/liter in aerobic fermentation. This was made possible by producing a mutant strain of a Type C Streptococcus zooepidemicus, HA-116, ATCC 39920, which is a high producer of hyaluronic acid and is haemolysin minus, i.e. of negligible pathogenicity. Aerobic Fermentation of S. zooepidemicus, HA-116, ATCC 39920 and subsequent purification of hyaluronate have resulted in batches of sodium hyaluronate with an average molecular weight of greater than 3.5.times.10.sup.6 daltons. This invention is the first method of producing and purifying such high molecular weight soidum hyaluronate by bacterial fermentation.
The first stage of the isolation procedure results in hyaluronic acid that is non-pyrogenic and non-irritating. This material can be further purified according to the methods of this invention to produce ultrapure, non-inflammatory hyaluronic acid suitable for clinical use.